A Study Of HPDGF-B Gene Modified Tissue-engineered Compound On Healing Of Periodontal Furcation Defects

Objective To transfect the plasmid(EX-A0380-M03) carried human platelet-derived growth factor-B(hPDGF-B) gene into gingival fibroblasts(GFs). To repair the experimental class II furcation defects of canine by a tissue-engineered compound which was constructed by transfected cells and acellular dermal matrix(ADM). To estimate the potential effects of hPDGF-B gene-modified GFs on periodontal regeneration and attain a new clew based on restoring the periodontal defects.Methods 1.The GFs of Beagle dog were isolated, cultured and purified by half-digestion and improved tissue block culture. The origination of tissue block was verified by operation and histology. Cells were identified by immunocytochemistry and detection of alkaline phosphatase. 2.The effects of two media, DMEM and RPMI1640, acting upon the GFs, were investigated by MTT, flow cytometry and immunocytochemistry. 3.The plasmid(EX-A0380-M03) was transformed, amplified and identified by a series of molecular biological methods.Subsequently, the plasmid identified correctly was transfected into GFs by liposome-mediated gene transfer method. RT-PCR, immunocytochemistry, ELISA as well as Western Blot were employed to observe the expression of hPDGF. 4.Morphological changes of transfected cells were recorded by optical and fluorescent microscope. Then, MTT and flow cytometry were used to estimate the proliferation and apoptosis of gene-modified cells.The expression of collagen I and XII, as well as theα-SMA was detected by Realtime Quantitative PCR. 5.GFs cultured on the surface of ADM were observed by histology and scanning electron microscope.The gene-modified tissue-engineered compound which was contructed by transfected GFs and ADM was implanted into subcutaneous tissue of nude mice. After 2, 4 or 8 weeks, animals were sacrificed and the regeneration of tissue-engineered compound was evaluated by histology. 6.Thirty experimental class II furcation defects in five Beagle dogs were prepared artificially.The ADM on which was seeded by transfected or non-transfected GFs at initial concentration of 5×106/mL was covered on the buccal side of experimental class II furcation defects.The animals were sacrificed at 12 weeks postoperatively and gingival regeneration was estimated by comparing the position of the gingival margin. Histological section prepared buccolingually was used to evaluate the regeneration of periodontal tissue.Results 1.The cells isolated was a kind of fibroblast-like cell originating from mesoderm. 2.There was no significant difference between DMEM and RPMI1640 in successful rate of emigration, proliferation and expression of collagen I. 3.Exogenous gene inserted in the plamid was hPDGF-B, which was confirmed by restriction enzyme digestion and DNA sequencing. Under the inverted-phase-contrast fluorescence microscope, cells emiting green fluorescence convinced the successful transfection,and the transfection efficiency was 18-38% by calculating the ratio of luminescent cells. RT-PCR, immunocytochemistry and ELISA convinced effective expression of hPDGF-B. Fusion protein(PDGF-BB—eGFP) expressed by transfected cells was detected by Western Blot. 4.Compared to the normal cells, morphological feature of partial transfected cells changed. Proliferation was enhanced after gene interference, but apoptosis has no obvious change.At transcription level, the expression of collagen I was up-regulated, while collagen XII andα-SMA was down-regulated. 5.After seeded on the ADM, GFs exhibited good growth. Under the subcutaneous tissue of nude mice, non-transfected group formed connective-like tissue, while transfected group formed osteoid at last.6.There was on significant difference between the transfected and non-transfected groups in gingival recession (P>0.05) after using the tissue-engineered compound to cure the class II furcation defects.However, the group using simple membrane(ADM and BME-10X) had worse recession(P<0.05) than two groups mentioned above. Buccal periodontal tissue regeneration of transfected group was the best one of the three experimental objects. Conclusion 1.Gingival fibroblasts were isolated and cultured successfully in vitro. 2.The two media, DMEM and RPMI1640 were both fit for the culture of gingival fibroblasts. 3.hPDGF-B was expressed efficiently in gingival fibroblasts. 4.The proliferation was increased after transfection and regeneration might be attained by collagen accumulation. 5. ADM exhibited excellent biocompatibility and was suitable for guided tissue regeneration as a kind of barrier membrane, also as a scaffold. 6.hPDGF-B gene enhanced tissue engineering might have a favorable application prospect in repairing the periodontal defects.